The ClpX protein of Bacillus subtilis indirectly influences RNA polymerase holoenzyme composition and directly stimulates σH-dependent transcription

In Bacillus subtilis, several processes associated with the onset of stationary phase, including the initiation of sporulation, require the activity of the minor sigma(H) form of RNA polymerase (RNAP). The induction of sigma(H)-dependent gene transcription requires the regulatory ATPase, ClpX. The ClpX-dependent post-exponential increase in sigma(H) activity is not dependent on the activator of sporulation gene expression, Spo0A. By determining the level of sigma(H) and sigma(A) in whole-cell extracts and RNAP preparations, evidence is presented that clpX does not influence the concentration of sigma subunits, but is required for the stationary phase reduction in sigma(A)-RNAP holoenzyme. This is probably an indirect consequence of ClpX activity, because the ClpX-dependent decrease in sigma(A)-RNAP concentration does not occur in a spo0A abrB mutant. The addition of ClpX to in vitro transcription reactions resulted in the stimulation of RNAP holoenzyme activity, but sigma(H)-RNAP was observed to be more sensitive to ClpX-dependent stimulation than sigma(A)-RNAP. No difference in transcriptional activity was observed in single-cycle in vitro transcription reactions, suggesting that ClpX acted at a step in transcription initiation after closed- and open-promoter complex formation. ClpX is proposed to function indirectly in the displacement of sigma(A) from core RNAP and to act directly in the stimulation of sigma(H)-dependent transcription in sporulating B. subtilis cells.